Wild Radish (Raphanus raphanistrum) is a dicot plant in the brassicaceae family. A single amino acid substitution from Proline 197 to Threonine has led to resistance to ALS inhibitors as indicated in the table below.

Acetolactate synthase (ALS)-inhibiting herbicide resistance is common in Raphanus raphanistrum (wild radish) populations across the Western Australian (WA) grain belt. This study investigates the molecular and biochemical basis of ALS herbicide resistance in five R. raphanistrum populations. Five known ALS herbicide resistance-endowing mutations (Pro-197-Ala, Pro-197-Thr, Pro-197-Ser, Asp-376-Glu and Trp-574-Leu) were identified, and their resistance spectrum to ALS-inhibiting herbicides was determined using purified populations individually homozygous for each mutation (except for Pro-197-Ala). Plants homozygous for ALS mutations at Pro-197 were found to be cross-resistant to ALS-inhibiting sulfonylurea (SU) and triazolopyrimidine (TP) herbicides, while plants homozygous for Trp-574-Leu were resistant to SU, TP and imidazolinone (IMI) ALS herbicide classes. The Asp-376-Glu mutation is reported here for the first time in R. raphanistrum populations and characterised at both the whole-plant and enzyme level. Plants homozygous for Asp-376-Glu were highly resistant to SU and TP herbicides, based on LD50 R/S ratios (>130 and 128 respectively) and I50 R/S ratios (170 and >110 respectively). In contrast, these plants were moderately resistant to the IMI imazamox (LD50 R/S ratio of 8, I50 R/S ratio of 3) and imazethapyr (I50 R/S ratio of 8) and susceptible to imazapyr (I50 R/S ratio of 0.76). A novel observation in this study is that resistance of homozygous Glu-376 plants is associated with a remarkable growth reduction in the presence of the ALS herbicides tested, making early resistance diagnosis and management difficult..

The biochemical and molecular basis of resistance to acetolactate synthase (ALS)-inhibiting herbicides was investigated in 8 resistant (R) and 3 susceptible (S) wild radish (Raphanus raphanistrum) populations. In vitro enzyme assays revealed an ALS herbicide-resistant ALS enzyme in all R populations. ALS enzyme extracted from the shoots of all eight R populations was highly resistant to the ALS-inhibiting sulfonylurea herbicide chlorsulfuron (20- to 160-fold) and the triazolopyrimidine herbicide metosulam (10- to 46-fold) and moderately resistant to metsulfuron (3 to 8-fold). There was little or no cross-resistance to the imidazolinone herbicides imazapyr and imazethapyr. The ALS gene fragment covering potential mutation sites in these populations was amplified, sequenced, and compared. All 8 R populations had point mutations in the codon for the proline residue in Domain A. However, the point mutations varied and encoded 4 different amino acid substitutions: histidine, threonine, alanine, and serine. No nucleotide difference in the DNA sequence of Domains C and D resulting in amino acid substitutions was observed between the R and S populations examined. In addition, a 3- to 5-fold higher ALS-specific activity was consistently observed in all R populations compared with S populations, whereas Northern blot analysis detected a similar level of ALS mRNA, suggesting a possible translational-posttranslational regulation of the enzyme. It is concluded that selection pressure from chlorsulfuron on eight separate wild radish populations has resulted in target site mutation at the same proline residue in the ALS gene. Higher ALS activity also may play a role in the resistance level..

The coding sequence of the entire acetolactate synthase (ALS) gene of wild radish (R. raphanistrum) was determined to be 1758 bp. Twelve ALS coding sequences were generated from representative populations collected from different geographical regions in Australia and consisted of populations that were either resistant or susceptible to ALS-inhibiting herbicides. Comparative analysis of the ALS gene sequences identified 81 base polymorphisms, 74 of which were neutral resulting in no amino acid change. There were seven loci where base changes resulted in amino acid substitutions in the gene. Five of these occurred randomly amongst the susceptible and resistant R. raphanistrum populations. Two of these five base polymorphisms were adjacent and accounted for three possible amino acids at the loci, indicating there are at least three alleles of the ALS gene in R. raphanistrum. The remaining two base mutations were observed only in the herbicide resistant populations. Three of the four resistant populations examined had a mutation, which disrupted the Pro codon in Domain A, a conserved region widely reported as conferring resistance to sulfonylurea herbicides in many weeds. The fourth herbicide resistant population had a mutation that caused a Trp->Leu substitution in Domain B. Studies have shown that this single point mutation endows a broad resistance to all four classes of ALS-inhibiting herbicides. An important aspect of our work was the development of molecular diagnostic assays for rapid screening of the pivotal mutations in Domains A and B in natural populations. Not every plant in the resistant, natural populations had the associated mutations, and some plants were heterozygotes for the resistant ALS gene allele characterized..